Defects in regulation of the prothrombinase complex play a central role in the pathogenesis of thrombosis. Activated protein C (APC) regulates the prothrombinese complex by inactivating factor V which serves as an essential protein cofactor. Resistance to activated protein C (APC) is the most common inherited risk factor for thrombosis. APC resistance is caused by a factor V mutation which blocks one of the three APC cleavage sites in the activated cofactor. The molecular mechanisms contributing to the normal and abnormal regulation of the prothrombinase complex by APC have not been completely elucidated. We have undertaken a systematic approach to understanding the functional importance of each of the three APC cleavage sites in factor Va and the mechanism for APC inactivation of factor Va. We have expressed and isolated factor V mutants in which cleavage at one two or all three APC cleavage sites is blocked. Our preliminary studies indicate that these mutants will be invaluable tools for dissecting the molecular mechanisms for APC resistance. In this revised application we propose to use our recombinant factor V expression system to further define the regulation of the prothrombinase complex by the protein C pathway. First, we will define the role of each individual APC cleavage site in the inactivation of factor V in both purified and plasma based systems. Second, we will characterize the mechanisms by which phosphatidylserine and phosphatidylethanolamine promote inactivation of the cofactor on synthetic and natural membranes. Third, we will use biochemical, immunological and molecular approaches to define the binding sites on factor Va for protein C. Finally, we will use factor V mutants that are resistant to both thrombin and APC to determine the nature and importance of the APC cofactor activity expressed by the procofactor, factor V. The results of these studies will define the precise molecular mechanisms that regulate inactivation of the prothrombinase complex by APC. This information will provide important insights into the pathophysiology of APC resistance and thrombosis and may ultimately lead to novel strategies for antithrombotic therapy.